PspA was the first surface protein of Streptococcus pneumoniae shown to elicit protection in mice against fatal sepsis, bacteremia, focal pneumonia, and colonization. It was also the first pneumococcal protein included in FDA-approved safety (phase 1) trials (1998, 2002, 2010 and 2012). These trials revealed no safety problems, and the human antibody elicited to PspA protected mice from fatal sepsis. All human trials with rPspA 1998, 2002, and 2010) examined (protection elicited by the ~300 aa alpha-helical domain (?HD) at the N-terminal end of the molecule. The ?HD is variable and up to three different ?HD may be required for a vaccine to cover the diversity of PspA in all pneumococci. We have recently shown that the ~100 aa proline-rich domain (PRD) can also elicit protection and two MAb to two different epitopes in the PRD can passively protect mice. The PRD domain is found in all PspAs and contains many shared 6-10 aa repeat sequences, at least one of which we already know encodes a linear protection-eliciting. If, at least a minority of the repeat sequences is immunogenic, it should be possible to create broadly cross-reactive PRD immunogens once the protective immunogenic epitopes are identified and combined. The delay and the enthusiasm for moving forward with clinical testing of protein vaccines are due, respectively, to the success and the problems (including strain replacement and high vaccine cost) associated with the polysaccharide-conjugate vaccines (PCVs). The development of protein vaccines is hampered by 1) the high cost and complexity of phase 3 trials in the presence of PCV usage; 2) the absence of data that the protein vaccines will protect against most of the human invasive disease (IPD) strains, which are not mouse-virulent and thus, cannot be tested in mice; and 3) the lack of a useful in vitro surrogate of protection to evaluate immunity to protein vaccines. Our proposal focuses on eliminating impediments to testing PspA in phase 2 and 3 trials. Aim 1 will validate our quantitative and scalable in vitro functional assays for protective antibody to PspA and determine whether a functional assay can measure protection against all human IPD isolates. Aim 2 will characterization protection-eliciting epitopes in the PRD and the develop optimal PRD immunogens that are optimally cross-protective. Aim 3 will compare optimal PRD and ?HD immunogens and determine if both are needed in a vaccine and whether PRD by itself can elicit protection against virtually all S. pneumoniae. The functional assays validated in Aim 1 will be important for the optimization of the PRD immunogen and for comparison of PRD versus ?HD. The functional assays will also be critical to evaluate protective responses in phase 1 and 2 trials to be able to provide some prediction about whether the expensive phase 3 trails are likely to be efficacious against Sp. Our functional assays may also aid the evaluation of vaccines containing more than one protection-eliciting pneumococcal surface antigen.